Difference between revisions of "Part:BBa K2680550"

(→‎Results)
(→‎Results)
Line 46: Line 46:
 
</html>
 
</html>
 
Group2：Co-expression
 
Group2：Co-expression
−
左边表格，右边图片
+
<html>
 +
<div style="display: flex;">
 +
    <table border="1" cellspacing="0" cellpadding="0">
 +
        <thead>
 +
            <td>ClpXP [nM]</td>
 +
            <td>eGFP degradation rate [nM/min]</td>
 +
        </thead>
 +
        <tr>
 +
            <td>0</td>
 +
            <td>9.3</td>
 +
        </tr>
 +
        <tr>
 +
            <td>0.2</td>
 +
            <td>29.4</td>
 +
        </tr>
 +
        <tr>
 +
            <td>0.4</td>
 +
            <td>47.3</td>
 +
        </tr>
 +
        <tr>
 +
            <td>1</td>
 +
            <td>80.1</td>
 +
        </tr>
 +
        <tr>
 +
            <td>2</td>
 +
            <td>128.9</td>
 +
        </tr>
 +
        <tr>
 +
            <td>6</td>
 +
            <td>128.4</td>
 +
        </tr>
 +
    </table>
 +
<img src="https://static.igem.org/mediawiki/parts/5/50/T--ZJUT-China--co_expression.png" width="200" style="margin-top: 50px;">
 +
</div>
 +
</html>
  
 
Degradation rate of deGFP upon different expression methods of clpXP. The degradation rate of deGFP-ssra only using the endogenous clpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-GFP-ssrA (5μM) to the cell-free system.
 
Degradation rate of deGFP upon different expression methods of clpXP. The degradation rate of deGFP-ssra only using the endogenous clpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-GFP-ssrA (5μM) to the cell-free system.

Revision as of 13:32, 10 October 2021

Contribution by Team ZJUT-China 2021

Group: Team ZJUT-China 2021
Author: Lianjie Sha and Xia Yao
Summary: According to the lectures, we learned that the degradation rate of eGFP-ssrA could be measured. In cell-free system, protein degradation by clpXP is described by a zeroth order chemical kinetic,and clpxp protein can recognize the protein with ssrA tag, so it is useful to add clpxp degrading the degfp. This year,on the basis work of iGEM18_William_and_Mary, ZJUT-China measured the different eGFP degradation rate by adding different concentrate of plasmid P70-clpxp (Part:BBa_K3885203) based on the reference. We hope it will support more help on modelling and further experiments.

Methodology

There are two methods to express clpxp protein: co-expression and pre-expression.Accelerated protein degradation can be achieved by co-expression of P70a-ClpXP, by adding protein to a cell-free system pre-incubated with P70a-ClpXP for an hour or by adding dilutions of pre-expressed clpXP (P70a-clpXP, 3nM). Different methods can provide different rates of protein degradation, ranging from 9.3 nM/min to 250 nM/min. By expressing clpXP, protein synthesis can be adjusted to an appropriate rate.[1]

Results

Group1: Pre-expression for an hour

ClpXP [nM] eGFP degradation rate [nM/min]
0 6.51±1.25
0.2 28.04±3.87
0.4 48.24±8.06
1 88.32±17.71
2 159.13±21.92
6 256.07±38.24
Group2：Co-expression
ClpXP [nM] eGFP degradation rate [nM/min]
0 9.3
0.2 29.4
0.4 47.3
1 80.1
2 128.9
6 128.4

Degradation rate of deGFP upon different expression methods of clpXP. The degradation rate of deGFP-ssra only using the endogenous clpXP has been determined by an assay, which is achieved by measuring the kinetics after adding pure His-GFP-ssrA (5μM) to the cell-free system.

Analysis

As shown in the figure above, the higher the concentration of CLPXP, the faster the degradation of eGFP-ssrA. Meanwhile, according to the table, when ClpXP with the same concentration was added, eGFP degradation rate in pre-expression was from 6.5 nM/min to 256 nM/min, while in co-expression, eGFP degradation rate was from 9.3 nM/min to 128 nM/min. It can be concluded that pre-expression is more conducive to eGFP protein degradation than co-expression.

Reference

[1] Garamella J, Marshall R, Rustad M, et al. The all E. coli TX-TL toolbox 2.0: a platform for cell-free synthetic biology[J]. ACS synthetic biology, 2016, 5(4): 344-355.